Numb was overexpressed in BM-MSCs (BM-MSCsNumb-OE) by cloning and transfecting the Numb gene. Lentiviral vectors (LV) were labeled with enhanced green fluorescent protein (EGFP). LV-Numb-RNA (titer: 3 × 108 TU/ml, Shanghai Genechem Co., Ltd., Shanghai, China) was transfected into P3 BM-MSCs at a multiplicity of infection (MOI) = 80 with the addition of both polybrene and enhanced infection solution (ENi. S, Shanghai GeneChem Co., Ltd., Shanghai, China). The component sequence of LV-Numb-RNA (20910-4) is Ubi-MCS-3FLAG-SV40-EGFP-IRES-puromycin, and its target sequence is shown in Additional file 1 : Text 1. The control BM-MSCs (BM-MSCoverexpression-empty vector, BM-MSCOE-EV) were transfected with CON238, an empty lentivirus vector (titer: 1 × 109 TU/ml, Shanghai Genechem Co., Ltd.), whose component sequence is Ubi-MCS-SV40-EGFP-IRES-puromycin. After transfection for 8–10 h, the medium was replaced with vector-free medium. Next, the cells were transfected with the lentivirus at an MOI = 80.
Con238
Manufactured by Genechem
CON238 is a laboratory centrifuge designed for general-purpose applications. It features a fixed-angle rotor that can accommodate a variety of sample tubes and microplates. The centrifuge provides consistent and reliable performance to support common laboratory procedures.
Automatically generated - may contain errors
Lab products found in correlation
Ubi mcs 3flag sv40 egfp ires puromycin, by Genechem (2 mentions)
Eni s, by Genechem (1 mentions)
Con077, by Genechem (1 mentions)
Hepg2, by American Type Culture Collection (1 mentions)
Fetal bovine serum (fbs), by Sartorius (1 mentions)
Hepg2, by Keygen Biotech (1 mentions)
Penicillin streptomycin, by Keygen Biotech (1 mentions)
Con335, by Genechem (1 mentions)
3 protocols using con238
1
Numb Overexpression in Bone Marrow-Derived MSCs
2
Lentiviral Transduction of A549 and H1299 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The lentiviral vectorsGV358‐RBM10 (14297‐1; Ubi‐MCS‐3FLAG‐SV40‐EGFP‐IRES‐puromycin) and GV248‐RBM10‐RNAi (66648‐1;hU6‐shTDP‐43‐Ubi‐EGFP‐IRES‐puromycin) and the corresponding control lentiviruses, CON238 and CON077, respectively, were obtained from Genechem (Shanghai, China). A549 cells and H1299 cells were infected with these lentiviral vectors. A total of 5 × 105 A549 cells and H1299 cells was seeded in a six‐well cell plate and further incubated for 12 hours to reach 30% confluency. A549 cells were infected with the lentiviral vectors at a multiplicity of infection (MOI) of 20 plaque‐forming units (PFU) per cell and H1299 cells at a MOI of 5 PFU per cell. The plates were then incubated for 24 hours prior to having their media changed to fresh, virus‐free media. Three days later, the GFP density was examined to evaluate the lentiviral infection efficiency.
3
Generating Stable HepG2 Cell Lines for ADAMTS16 and BMP2 Overexpression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The human hepatoblastoma cell line, HepG2, was purchased from ATCC via the Shanghai Huiying Biological Technology Co., Ltd. The cell line was authenticated by human STR profiling and confirmed to be mycoplasma free. HepG2 cells were cultured in DMEM (Nanjing KeyGen Biotech Co., Ltd.) containing 10% fetal bovine serum (FBS; Biological Industries) and 1% penicillin-streptomycin (Nanjing KeyGen Biotech Co., Ltd.), and incubated in a humidified 5% CO2 incubator at 37°C.
The lentiviral vectors, GV513-ADAMTS16 (Ubi-MCS-CBh-gcGFP-IRES-puromycin) and GV358-BMP2 (Ubi-MCS-3FLAG-SV40-EGFP-IRES-puromycin), and the corresponding control lentiviruses containing the empty vector, CON335 and CON238, respectively, were purchased from Shanghai GeneChem Co., Ltd. Accession numbers for the two genes used in the present study are as follows: ADAMTS16, NM139056; BMP2, NM001200. For lentiviral infection, HepG2 cells were incubated with lentivirus at a MOI of 10 for 16 h, and the stable cell line was selected using 2 µg/ml puromycin for a week, followed by 2 µg/ml puromycin maintenance. The overexpression efficiency of ADAMTS16 and BMP2 were assessed through reverse transcription-quantitative PCR (RT-qPCR).
The lentiviral vectors, GV513-ADAMTS16 (Ubi-MCS-CBh-gcGFP-IRES-puromycin) and GV358-BMP2 (Ubi-MCS-3FLAG-SV40-EGFP-IRES-puromycin), and the corresponding control lentiviruses containing the empty vector, CON335 and CON238, respectively, were purchased from Shanghai GeneChem Co., Ltd. Accession numbers for the two genes used in the present study are as follows: ADAMTS16, NM139056; BMP2, NM001200. For lentiviral infection, HepG2 cells were incubated with lentivirus at a MOI of 10 for 16 h, and the stable cell line was selected using 2 µg/ml puromycin for a week, followed by 2 µg/ml puromycin maintenance. The overexpression efficiency of ADAMTS16 and BMP2 were assessed through reverse transcription-quantitative PCR (RT-qPCR).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!